Cleavage of delta and epsilon tubulin PCR products is observed upon PCR purification

In this study, we report an unusual phenomenon of the self-cleavage of purified PCR products of codon-optimized Chlamydomonas reinhardtii delta tubulin ( uni3 ) and epsilon tubulin ( bld2 ) genes through an unknown mechanism. Our studies revealed that intact PCR products for both these genes could be obtained upon PCR amplification from plasmid templates carrying these genes. However, interestingly, purification of these PCR products led to their cleavage through an unidentified mechanism. This cleavage persisted despite using different PCR purification kits. Deleting a synthetic intron within the delta tubulin gene also did not have any effect on this cleavage.


Figure 1.
Full-length PCR products of codon-optimized Chlamydomonas reinhardtii delta and epsilon tubulin genes were successfully obtained upon PCR.However, PCR purification of these PCR products led to their cleavage through an unknown mechanism.A) Agarose gel electrophoresis of delta tubulin (uni3), epsilon tubulin (bld2) and positive control (dyn-1) PCR products showing that PCR products of the expected sizes of 1775 bp for delta tubulin (uni3), 1874 bp for epsilon tubulin (bld2) and 1011 bp for the positive control (dyn-1) were obtained.B) Purification of the delta and epsilon tubulin PCR products using either the Invitrogen Purelink™ Quick PCR purification kit (Thermo Fischer Scientific, Waltham, MA) or the Qiagen Qiaquick® PCR purification kit (Qiagen, Redwood City, CA) led to cleavage of the PCR products as seen by the appearance of lower sized bands between 650 bp and 1000 bp upon agarose gel electrophoresis (red stars).Importantly, the PCR product of the positive control (dyn-1) behaved as expected and did not exhibit cleavage upon purification with either kit.C) Agarose gel electrophoresis showing that the deletion of a synthetic intron close to the identified cleavage junction within the sequence of delta tubulin did not eliminate cleavage of the delta tubulin PCR product upon PCR purification with either kit (red stars).

Description
Centrosomes are the major microtubule organizing centers of most animal cells.They contribute to a variety of cellular functions including spindle assembly, cell polarity, cell shape and cilia assembly (Reviewed in Nigg et al., 2009;Bornens et al., 2012;Conduit et al., 2015;Fu et al., 2015;Nigg and Holland, 2018).Centrosomes consist of orthogonally-paired cylindrical centrioles enveloped by the protein-dense pericentriolar material (Reviewed in Schwarz et al., 2018).Chlamydomonas reinhardtii delta and epsilon tubulin proteins that are coded by the uni3 and bld2 genes respectively, play a crucial role in maintaining the proper triplet microtubule architecture of centrioles (Dutcher and Trabuco, 1998;Dutcher et al., 2002).To obtain further insights into the functions of these genes in regulating centriole architecture, we wanted to generate purified PCR products of Chlamydomonas reinhardtii delta and epsilon tubulin cDNAs carrying synthetic introns that were codon-optimized for expression in C. elegans.These purified PCR products would enable us to perform downstream functional assays to further characterize the functions of these genes.Although we were successful in obtaining full length PCR products of codon-optimized Chlamydomonas reinhardtii delta and epsilon tubulin genes (Figure 1A), these PCR products exhibited an unexpected cleavage upon their purification using either the Invitrogen Purelink™ Quick PCR purification kit or the Qiaquick® PCR purification kit (Figure 1B).As shown in Figure 1B, both the delta and epsilon tubulin PCR products but not a non-relevant control PCR product (dyn-1) exhibited cleavage after their purification using the Invitrogen Purelink™ Quick PCR purification kit or the Qiaquick® PCR purification kit.Purification of the delta and epsilon tubulin PCR products using a non-kit-based isopropanol precipitation method as well as upon their gel extraction using a Qiaquick Gel Extraction kit (Qiagen, Redwood City, CA), both yielded this similar pattern of cleavage.To confirm that the cleaved fragments indeed correspond to the PCR products of the delta and epsilon tubulin genes, DNA sequencing of these lower sized fragments was performed after excising these separated bands from the agarose gel.Our sequencing results confirmed that the lower sized fragments that were observed between 650 bp and 1000 bp upon agarose gel electrophoresis of the purified delta and epsilon tubulin PCR products (Figure 1B) contained DNA sequences from these respective genes.Our sequencing analysis identified a synthetic intron close to the genomic region where the delta and epsilon tubulin PCR products were being cleaved.Since the sequence of this synthetic intron was identical between the delta and epsilon tubulin genes, we questioned whether the presence of this synthetic intron was responsible for the cleavage of the purified PCR products of these genes.To address this, site-directed mutagenesis was performed to remove this synthetic intron from the sequence of the delta tubulin gene and the PCR purification procedure was repeated.As shown in Figure 1C, deletion of the synthetic intron close to the cleavage region within the delta tubulin gene did not eliminate cleavage of the purified PCR product.These data indicate that this synthetic intron does not contribute to this observed cleavage phenomenon.In the future, studies should be directed to further characterize the molecular mechanisms mediating the cleavage of the Chlamydomonas reinhardtii delta and epsilon tubulin PCR products upon purification.Since we have invested a considerable amount of time and resources on this project, we would like to share these findings with the scientific community and with other researchers working on these genes to prevent a further loss of their valuable time and resources.

Methods
I) PCR conditions, analysis, and purification PCR was performed using a C1000™ Thermal Cycler (BioRad, Hercules, CA).35 cycles of amplification were used for 50 µL PCR reaction volume consisting of: 1 µL-10 µM forward primer, 1 µL-10 µM reverse primer, 35 ng-plasmid template (uni3, bld2, dyn-1), 1 µL -10 mM dNTPs, 2 µL-50 mM MgSO 4 , 5 µL -10X Hi-Fi Buffer, 0.2 µL -Platinum® Taq DNA Polymerase Hi -Fi enzyme, and molecular biology water was used to bring the final volume to 50 µL.To verify the results of PCR amplification, the PCR products were analyzed by gel electrophoresis on a 1% agarose gel.The PCR-amplified products were further purified using Invitrogen Purelink™ Quick PCR purification kit and Qiaquick® PCR purification kit.Note that molecular biology grade water was used to elute the PCR products in the final step for PCR purification using both these kits instead of the elution buffer.Upon elution, the purified PCR products were analyzed by gel electrophoresis on a 1% agarose gel.

II) Site-directed mutagenesis
For performing the site-directed mutagenesis, the PCR reaction mixture comprised of all the same components used in PCR amplification of the uni3 gene except that the Phusion® High-Fidelity DNA Polymerase was used for PCR amplification.
PCR conditions for delta tubulin intron deletion using site-directed mutagenesis: Initial denaturation: 98°C -30 s Denaturation: 98°C -10 s Annealing: 68.6°C -30 s Extension: 72°C -6 min Final Extension: 72°C -10 min Site-directed mutagenesis was followed by digestion with 5 units of Dpn1 for 2 hours, followed by bacterial transformation and plasmid purification using the QIAprep® Spin Miniprep kit.The isolated plasmid samples were sent for whole plasmid sequencing (Plasmidsaurus LLC, Eugene, OR).Upon analyzing the sequencing results, the plasmid with successful intron deletion was selected and used for further analysis.
Sequence of the positive control used in this study (dyn-1):